March 2014355
Regular Article
Biol. Pharm. Bull. 37(3) 355–360 (2014)

Monitoring of Geﬁtinib Sensitivity with Radioiodinated PHY Based on
EGFR Expression
Mitsuyoshi Yoshimoto,*,a,b Masahiko Hirata,c Yasukazu Kanai,d Sadahiro Naka,e Ryuichi Nishii, fShinya Kagawa,g Keiichi Kawai,b and Yoshiro OhmomocaDivision of Functional Imaging, National Cancer Center Hospital East; 6–5–1 Kashiwanoha, Kashiwa, Chiba
277–8577, Japan: b Faculty of Health Sciences, Kanazawa University; 5–11–80 Kodatsuno, Kanazawa, Ishikawa
920–0942, Japan: c Osaka University of Pharmaceutical Sciences; 4–20–1 Nasahara, Takatsuki, Osaka 569–1094,
Japan: d Department of Molecular Imaging in Medicine, Osaka University Graduate School of Medicine; e Departmentof Nuclear Medicine and Tracer Kinetics, Osaka University Graduate School of Medicine; 2–2 Yamadaoka, Suita,
Osaka 565–0871, Japan: f Department of Radiology, Faculty of Medicine, University of Miyazaki; 5200 Kihara,
Kiyotake-cho, Miyazaki 889–1692, Japan: and g Research Institute, Shiga Medical Center; S 5–4–30 Moriyama,Moriyama, Shiga 524–8524, Japan.
Received July 16, 2013; accepted December 10, 2013
Epidermal growth factor receptor (EGFR) is attractive target for tumor diagnosis and therapy, as it isspeciﬁcally and abundantly expressed in tumor cells. EGFR-tyrosine kinase (TK) inhibitors such as geﬁtinib
and erlotinib are widely used in the treatment of non-small cell lung cancer (NSCLC). In this study, weinvestigated whether radioiodinated 4-(3-iodo-phenoxy)-6,7-diethoxy-quinazoline (PHY), which is a candidate EGFR-TK imaging agent for single photon emission computed tomography (SPECT) is able to predict
geﬁtinib sensitivity. We used four NSCLC cell lines-A549 (wild-type EGFR), H1650 (mutant EGFR; delE746_A750), H1975 (mutant EGFR; L858R, T790M) and H3255 (mutant EGFR; L858R)-and one epidermoid
carcinoma cell line, A431 (wild-type EGFR). Cell proliferation assay and Western blotting revealed that
A431 and H3255 with high EGFR expression showed high sensitivity to geﬁtinib. On the other hand, A549,
H1650 and H1975 showed much lower sensitivity to geﬁtinib. The blocking study revealed that geﬁtinib decreased tumor uptake in 125I-PHY in A431-bearing mice. Moreover, in vivo tumor uptake of 125I-PHY wascorrelated with the IC50 of geﬁtinib for cell proliferation. In the present study, tumor uptake of 125I-PHY
was correlated with the geﬁtinib sensitivity and this uptake was based on expression levels of EGFR, but
not on mutation status. Although the mutation status is the most important factor for predicting geﬁtinib
sensitivity, the abundant expression of EGFR is essential for therapy with EGFR-TK inhibitors. Therefore,
radioiodinated PHY is a potential imaging agent to predict geﬁtinib sensitivity based on EGFR expression
levels though further modiﬁcations of the imaging agent is needed to accurately estimate the mutation status.
Key words tumor imaging; geﬁtinib; epidermal growth factor receptor tyrosine kinase; lung cancer; single
photon emission computed tomography

Epidermal growth factor receptor (EGFR) is overexpressedin a variety of tumors including non-small cell lung cancer(NSCLC), head and neck cancer, glioma and ovarian cancer.1,2)
Activation of EGFR tyrosine kinase (EGFR-TK) is involved in
the proliferation, invasion, metastasis, angiogenesis and suppression of apoptosis.3) Therefore, EGFR is an attractive target
molecule for tumor diagnosis and therapy.
To date, numerous EGFR-TK inhibitors and anti-EGFR
antibodies have been developed and used in clinical settings.2,4) Geﬁtinib, an EGFR-TK inhibitor, has been approved
as a molecular targeted agent for the treatment of advanced
NSCLC. Geﬁtinib leads to rapid symptom improvement andtumor regression in patients with NSCLC. Tumor responses
were, however, observed in only 10–19% of patients with
NSCLC in the phase II trial.5,6) In addition, interstitial lungdisease is a serious and fatal adverse effect of geﬁtinib treatment.7) Retrospective epidemiologic analyses have revealed
that the therapeutic effects are more apparent in Japanese,
women and non-smokers.8) From recent molecular biologicalstudies,9–11) mutations in the TK domain of EGFR gene areclosely associated with geﬁtinib sensitivity. Therefore, development of diagnostic technology is necessary to predict or
estimate therapeutic efﬁcacy by EGFR-TK inhibitors.

Nuclear medicine techniques (positron emission tomography; PET and single photon emission computed tomography;
SPECT) are able to estimate physiological and biological
functions at the molecular or gene levels, as well as pharmacokinetics in vivo.12,13) Radiolabeled inhibitors are believed
to clarify pharmacokinetics, expression levels and functionsof target molecules, and to estimate the therapeutic efﬁcacyof the inhibitors. Although 18F-geﬁtinib was investigated tomonitor EGFR expression and mutation status, tumor uptake
was not correlated with EGFR expression levels and mutation
status.14) Development of various EGFR-TK imaging agentsbased exclusively on the anilinoquinazoline structure has been
attempted for more than a decade, but further optimization of
the properties of imaging agents with respect to high speciﬁcity to EGFR, selectivity to mutant types of kinase and hightumor uptake is required.15–20) Thus, novel imaging agents toaccurately detect EGFR-TK activity are needed in order topredict and monitor the therapeutic effects.
We have reported that radioiodinated 4-(3-iodo-phenoxy)6,7-diethoxy-quinazoline (PHY, Fig. 1) is a candidate EGFRTK imaging agent for SPECT.21) In this report, we measured
the uptake of 125I-PHY in tumors with various expression levels of EGFR and types of mutation, and investigated whether
geﬁtinib sensitivity can be estimated with radioiodinated PHY.

The authors declare no conflict of interest.
* To whom correspondence should be addressed. e-mail: miyoshim@ncc.go.jp© 2014 The Pharmaceutical Society of Japan

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Fig. 1.

Table 1.

Vol. 37, No. 3

Chemical Structures of 4-(3-Iodo-phenoxy)-6,7-diethoxy-quinazoline (PHY), PD153035 and Geﬁtinib

Characterization and Sensitivity to Geﬁtinib in Tumor Cell Lines Used in This Study
Cell lines

Origin

EGFR status

IC50 (μM)

A431
A549
H1650
H1975
H3255

Human epidermoid carcinoma
Human NSCLC (Adenocarcinoma)
Human NSCLC (Adenocarcinoma)
Human NSCLC (Adenocarcinoma)
Human NSCLC (Adenocarcinoma)

Wild-typeWild-typedel E746-A750
L858R, T790M
L858R

1.8
17.4
12.1
13.3
0.02

MATERIALS AND METHODS
Cell Culture Four NSCLC cell lines-A549 (wild-type
EGFR), H1650 (mutant EGFR; del E746_A750), H1975 (mutant EGFR; L858R, T790M) and H3255 (mutant EGFR;
L858R)-and one epidermoid carcinoma cell line, A431 (wildtype EGFR), were used in this study (Table 1). A431 and A549
were obtained from DS Pharma Biomedical (Osaka, Japan).
H1650 and H1975 were purchased from the American Tissue Culture Collection (Manassas, VA , U.S.A.). H3255 was
kindly provided by Dr. Juri Gelovani (M.D. Anderson Cancer
Center). A431 and A549 were maintained in Dulbecco’s modiﬁed Eagle’s medium (DMEM; Sigma, St. Louis, MO, U.S.A.)
supplemented with 10% fetal bovine serum (FBS; InvitrogenInc., Carlsbad, CA, U.S.A.). H1650 and H1975 were maintained in RPMI1640 (Sigma) supplemented with 10% FBS
and 2 m M glutamine. H3255 was maintained in DMEM/F12(Sigma) supplemented with 20% FBS. All cells were cultured
in a 5% CO2-humidiﬁed atmosphere at 37°C.
Growth Inhibition Assay Growth inhibition by geﬁtinib
was assessed using CellTiter 96® AQueous One Solution Cell
Proliferation Assay Kit (Promega, Madison, WI, U.S.A.).
Cells were seeded in 96-well plates and incubated in culture
media overnight at 37°C. The numbers of cells per well usedin these experiments were as follows: 4000 for A431; 1000
for A549; 3000 for H1650; 2500 for H1975; and 5000 for
H3255. After incubation with geﬁtinib, PD153035 and PHY
at concentrations ranging from 1 n M to 33.3 µM for 72 h, 20 µL
of CellTiter 96® AQueous One Solution was added to each welland plates were incubated for a further 2 h at 37°C. Absorbance was measured at 490 nm using a micro-plate reader.
Each experiment was set up in four replicate wells for eachdrug concentration and was repeated at least 4 times. IC50 values were determined using a nonlinear regression model witha sigmoidal dose response (GraphPad Prism version 4.00 for
Widows; GraphPad Software, San Diego, CA, U.S.A.).
Western Blotting Cells were lysed in radio immunoprecipitation assay (RIPA) buffer [50 m M Tris–HCl (pH 8.0),
150 m M NaCl, 1% NP-40, 0.5% deoxycholic acid sodium

salt, 0.1% sodium dodecyl sulfate (SDS)] containing proteaseinhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and phosphatase inhibitor cocktail (Nacalai Tesque). Protein concentration was determined using BCA Protein Assay Reagent kit(Thermo-Fisher Scientiﬁc, Rockford, IL, U.S.A.). Lysates wereseparated by SDS-polyacrylamide gel electrophoresis (PAGE)
and transferred onto a polyvinylidene difluoride (PVDF)
membrane (Immobilon-FL, Millipore, Billerica, MA, U.S.A.).
Membranes were blocked for 1 h in LI-COR blocking buffer
and were incubated with primary and secondary antibodies.
Rabbit polyclonal anti-EGFR (SC-03) (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), anti-phospho-EGFR (Tyr1068)(Cell Signaling Technology, Danvers, MA, U.S.A.) and mousemonoclonal anti-β actin (Sigma) were used as primary antibodies. Alexa Fluor 680 goat anti-rabbit immunoglobulin G(IgG) (Molecular Probes, Eugene, OR, U.S.A.) and IRDye 800
anti-mouse IgG (Rockland Immunochemicals, Gilbertsville,
PA, U.S.A.) were used as secondary antibodies. Membranes
were scanned and bands were detected using the Odyssey
Infrared Imaging System (LI-COR Biosciences, Lincoln, NE,
U.S.A.).
Tumor Xenograft Model Animal studies were performedin compliance with the guidelines for the care and use oflaboratory animals of Kanazawa University. Biodistribution
studies were conducted in nude mice bearing tumors. Female BALB/c nu/nu mice aged 5–6 weeks (Japan SLC, Inc.
Hamamatsu, Japan) were xenografted s.c. in the right and left
dorsum with 2–5×106 cells. Tumor size was below 1 cm indiameter.
Radiosynthesis of 125I-PHY 125I-NaI (MP Biomedicals,
Santa Ana, CA, U.S.A.) and 10 µL of 0.1 N HCl were added
to 20 µL of tributylstannyl precursor (1 mg/mL in EtOH), followed by addition of 10 µL of 30% hydrogen peroxide. After
mixing for 5 min, the reaction was quenched by adding 10 µL
of 10% sodium metabisulfate. Puriﬁcation of 125I-PHY was
performed by HPLC using a Cosmosil 5C18-AR II column(10×250 mm; Nacalai Tesque) eluted with 85% MeOH and
15% water at a flow rate of 2.0 mL/min.
Biodistribution of 125I-PHY in Tumor-Bearing Mice

March 2014357

Tumor-bearing mice were injected via the tail vein with
74 kBq/100 µL (saline) of 125I-PHY. Mice were sacriﬁced at
1, 4 and 24 h post-injection, and tissue samples were excised.
To investigate EGFR-TK-speciﬁc uptake of 125I-PHY, geﬁtinib(10 mg/kg) was co-injected with 125I-PHY. Biodistribution wasdetermined at 4 h post-injection. Tissue samples were weighed
and radioactivity was measured with a γ-counter (ARC-360,
Aloka, Tokyo, Japan). Uptake in organs was expressed as thepercentage of injected dose per gram of tissue (%ID/g).
Statistical Analysis Data analysis was performed using
GraphPad Prism. Unpaired t-test was used for the blocking
study. The correlation between IC50 of geﬁtinib and tumor uptake of 125I-PHY was tested using Spearman correlation test.
The results were considered statistically signiﬁcant at p<0.05.

RESULTS
Expression of EGFR and Phospho-EGFR and Growth
Inhibition by Geﬁtinib We used ﬁve tumor cell lines (2wild-type EGFR and 3 mutant EGFR) and analyzed EGFR
and phosphorylated EGFR (phospho-EGFR) expression levels
by western blotting (Fig. 2). IC50 values for growth inhibition by geﬁtinib were also measured by MTS assay (Table 1).
There was high expression of EGFR and phospho-EGFR in
A431 and H3255, whereas the IC50 of H3255 (L858R) (0.02 µM)
was much lower than that of A431 (1.8 µM). The highest IC50
was observed in A549 (17.4 µM), although the expression ofFig. 2. Expression of EGFR and Phospho-EGFR, as Detected by Western Blotting

Fig. 3.

EGFR and phospho-EGFR was moderate. H1650 (del E746A750) and H1975 (L858R+T790M) showed lower expressionof EGFR and phospho-EGFR, and the IC50 values for H1650
and H1975 were 12.1 µM and 13.3 µM, respectively.
Effect of L858R Mutation on Growth Inhibition by
EGFR Inhibitors In order to investigate whether EGFR
mutations affect growth inhibition by PHY and geﬁtinib,
A431 and H3255 were selected because they have abundant
expression of EGFR and show different geﬁtinib sensitivity.
The IC50 of PD153035, which is an analogue of PHY, was alsomeasured to investigate the structure–activity relationship. All
Table 2. IC50 of Geﬁtinib, PD153035 and PHY against Cell Growth inA431 (Wild EGFR) and H3255 (L858R)

Geﬁtinib (µM)
PD153035 (µM)
PHY (µM)
Table 3.

H3255

A431/H3255

1.80
1.63
6.50

0.02
0.29
2.10

90.0
5.6
3.1

Biodistribution of 125I-PHY in A431-Bearing Nude Mice (n=4)

Blood
Brain
Liver
Heart
Lung
StomachKidneyPancreas
SpleenMuscleThyroid (%ID)
Intestine
A431

1h

4h

24 h

0.72±0.06
0.24±0.02
3.30±1.15
0.65±0.10
1.08±0.17
4.60±1.82
1.33±0.16
0.84±0.13
0.52±0.10
1.12±0.39
0.27±0.08
30.57±6.07
1.56±0.25

0.62±0.09
0.06±0.01
1.62±0.78
0.27±0.04
0.51±0.04
2.90±1.64
0.62±0.06
0.31±0.07
0.22±0.04
0.23±0.01
0.68±0.13
22.70±5.14
0.47±0.12

0.03±0.01
0.00±0.00
0.07±0.02
0.02±0.01
0.06±0.04
0.13±0.08
0.03±0.01
0.01±0.01
0.02±0.02
0.05±0.06
0.82±0.13
0.92±0.55
0.03±0.01

Data are expressed as %ID/g (mean±S.D.).

Biodistribution of 125I-PHY at 4 h with and without Co-injection of Geﬁtinib (n=3–4)

Radioactivity in tissues is expressed as %ID/g (mean±S.D.). * p<0.05 vs. control.

A431

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Vol. 37, No. 3

compounds showed potent growth inhibition in H3255 (Table2). Increased inhibitory effects due to this mutation were only
observed in geﬁtinib treatment. The IC50 in H3255 was morethan 90-fold lower than that in A431, while the IC50 values
for PD153035 and PHY in H3255 were only 5.6- and 3.1-foldlower than those in A431.
Biodistribution of 125I-PHY in A431-Bearing Mice
Table 3 shows the biodistribution of 125I-PHY in A431-bearing
mice. 125I-PHY showed signiﬁcant uptake in the intestine(30.6%ID/g at 1 h) and moderate uptake in the liver and stomach (3.3%ID/g and 4.6%ID/g at 1 h, respectively). There was
low uptake of 125I-PHY in other normal tissues. 125I-PHY was
rapidly cleared, whereas only the uptake in thyroid slowly increased up to 24 h post-injection (0.27%ID at 1 h to 0.82%ID
at 24 h). Figure 3 indicates the effects of geﬁtinib on 125I-PHY
uptake at 4 h post-injection. Co-injection of geﬁtinib with
125
I-PHY resulted in a signiﬁcant decrease in uptake in A431
(0.50%ID/g for control and 0.26%ID/g for geﬁtinib blocking).
Relationship between Tumor Uptake of 125I-PHY andGeﬁtinib Sensitivity 125I-PHY accumulated at various levels
in tumors with different EGFR expression levels, as shown in
Fig. 4 (1.56%ID/g for A431, 1.04%ID/g for A549, 1.31%ID/g
for H1650, 0.94%ID/g for H1975 and 2.04%ID/g for H3255).
Tumor uptake was reduced to less than half at 4 h post-injection. Tumors to blood (T/B) ratio and tumor to muscle (T/M)
ratios were entirely good at 1 h post-injection (Fig. 5). A431,

H1650 and H3255 showed good tumor to lung (T/L) ratios.
As shown in Fig. 6, there is a signiﬁcant correlation between
125
I-PHY uptake and geﬁtinib sensitivity (IC50) (r=−0.900,
p<0.05).

DISCUSSION
We investigated the correlation between in vivo tumor uptake of 125I-PHY and geﬁtinib sensitivity. In this study, weused one epidermoid carcinoma cell line and four NSCLC celllines with different levels of EGFR expression and mutation
status. These cell lines showed a range of geﬁtinib sensitivities. The present study indicated that the radioiodinated PHY
is able to estimate geﬁtinib sensitivity based on EGFR expression levels.
Biodistribution studies indicated that 125I-PHY is signiﬁcantly taken up in the intestine, followed by the stomach andliver. This biodistribution was similar to that of other EGFRTK imaging agents.14,19) One preferable characteristic is that
125
I-PHY uptake in blood, lung and brain is low, resulting in
relatively high T/B and T/L ratios. This suggests that radioiodinated PHY is useful for imaging lung and brain tumors.
The in vivo tumor uptake of 125I-PHY is EGFR-TK speciﬁc
and is negatively correlated with the IC50 values of geﬁtinib in
ﬁve cell lines. These results are in accordance with the previ-

Fig. 6. Correlation between in Vivo Tumor Uptake at 1 h and Geﬁtinib
Sensitivity (r=−0.900, p<0.05)

Fig. 4.

In Vivo Uptake of 125I-PHY in Tumor Bearing Mice (n=4)

Fig. 5.

Tumor to Blood (T/B), Muscle (T/M) and Lung (T/L) Ratios of 125I-PHYMarch 2014359

ous results18) that indicate correlations between 11C-PD153035
uptake and EGFR expression levels, as measured by flow
cytometry. PHY is structurally similar to PD153035 and theinhibitory potency of PHY against EGFR-TK phosphorylationis the same as that of PD153035.
Some tumor uptake is caused by nonspeciﬁc binding of
125
I-PHY, most likely to the cell membrane. In A431 andA549 harboring wild-type EGFR, geﬁtinib sensitivity of A431
was 10-fold higher than that of A549 and western blotting
also showed abundant expression of EGFR in A431 whencompared with A549, while 125I-PHY uptake in A431 wasonly 1.5-fold higher than that in A549. An excess of geﬁtinib
reduced 125I-PHY uptake in A431 up to 52%. Therefore, reduction in nonspeciﬁc binding would result in estimating EGFR
expression in more detail.
Although radiolabeled geﬁtinib should be suitable for estimating geﬁtinib sensitivity and assessing mutation status,
18
F-geﬁtinib did not reflect EGFR expression levels.14) 125I-PHY
and 11C-PD153035 were washed out of tumors, whereas 18Fgeﬁtinib was retained in tumors throughout the experiment.
These observations suggest that nonspeciﬁc binding of 18Fgeﬁtinib to the cell membrane predominantly contributes totumor uptake. In comparison with geﬁtinib, therefore, PHY
and PD153035 may successfully penetrate into the cell membrane and then bind to EGFR-TK.
Estimation of EGFR expression would be a useful index
for selecting patients to beneﬁt from treatment with EGFR-TK
inhibitors and to monitor inhibition status. Several groups9–11)
have shown that mutations in the TK domain of the EGFR
gene are signiﬁcantly correlated with clinical response to geﬁtinib therapy. Therefore, this mutation is a candidate to predict
geﬁtinib sensitivity. Unfortunately, there are no signiﬁcant differences in tumor uptake and clearance of 125I-PHY between
cells with wild-type EGFR and mutant EGFR. However, theoverexpression is a prerequisite for treatment with EGFR-TK
inhibitors.
Imaging agents are required to detect mutation status for
accurate prediction of therapeutic efﬁcacy with EGFR-TK inhibitors. The level of EGFR expression in tumors is, in itself,
insufﬁcient to account for their sensitivity to treatment.22,23)
PHY and PD153035 showed slight enhancement of growth inhibition in H3255, with the IC50 values for PHY and PD153035
being 3.1- and 5.6-fold lower than those in A431, respectively.
In contrast, geﬁtinib showed marked growth inhibition in
H3255, in which the IC50 of geﬁtinib is 90-fold lower than in
A431. Crystal structure analysis24,25) revealed that substitutionof Leu858 with arginine maintains kinase activity in the active state and allows geﬁtinib to move deeper inside the binding pocket. Although 18F-geﬁtinib is unsuitable for imaging
EGFR-TK activity, these ﬁndings suggest that introductionof propylmorpholino or other substituents to the 6 position ofquinazoline is required for assessing mutation status.

CONCLUSION
Although EGFR expression levels are not sufﬁcient to predict treatment efﬁcacy with EGFR-TK inhibitors, EGFR overexpression in tumors is an essential factor for EGFR targeted
therapy. Our study suggested that radioiodinated PHY is apotential imaging agent to detect EGFR expression levels as aﬁrst screening marker and we require further modiﬁcations of

the compound to accurately estimate geﬁtinib sensitivity.
Acknowledgments This work was supported by Grantsin-Aid for Scientiﬁc Research (C) No.10770466, No.19591437,(B) No.18390331 from JSPS and a Grant-in-Aid for HighTechnology Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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